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Definitions

• the present invention relates to gene therapies for use in treating monogenic forms of nephrotic syndrome
• Nephrotic syndrome is a chronic kidney disease characterized by significant proteinuria, hypoalbuminemia, oedema and hyperlipidemia, and is the most common primary glomerular disease in children, affecting 2/100,000 children below the age of 16 in Europe and the USA.
• NS is associated with different ages of onset, from less than 3 months old at diagnosis to early adulthood, and is classified in different patients groups depending on their sensitivity to corticoids: ⁇ 80% of children with NS are classified as having steroid-sensitive nephrotic syndrome (SSNS) and can be successfully treated with corticosteroid therapy.
• SSNS steroid-sensitive nephrotic syndrome
• SRNS steroid- resistant nephrotic syndrome
• the podocyte branches off cellular processes to cover the outside of the glomerular capillary, called foot processes, and their interdigitations with neighbouring foot processes form the glomerular slit membrane, critical for the glomerular filtration barrier efficiency and for the retention of protein in the blood stream.
• mutations in genes coding for key podocyte processes such as the development, migration, basement membrane interaction, or regeneration of the podocyte, lead to the loss of integrity of the glomerular slit membrane and to the nephrotic syndrome phenotype.
• Approximately 30% of cases of SRNS in children are genetic, where the most common mutations in childhood are in NPHS2 encoding podocin, accounting for 10-30% of sporadic genetic cases.
• Podocin is a 42kDa hairpin like membrane-associated podocyte-specific protein that is a key component of the protein complex at the slit diaphragm; the cell-cell junction between adjacent podocyte foot processes. It localises to lipid rafts and interacts with other important slit diaphragm proteins like nephrin, CD2AP and TRPC6. It is essential in the maintenance of the slit diaphragm, and consequently the integrity of the glomerular filtration barrier. There are 126 mutations reported to date, but the most common mutation is R138Q, which causes mislocalisation of podocin to the endoplasmic reticulum.
• tail vein injection was shown to be unsuitable for kidney transduction and, while a low level of gene expression in podocytes was observed, widespread expression was also observed in the liver, despite the use of an allegedly kidney specific promoter.
• the study additionally failed to demonstrate successful transduction of a NS-associated transgene, such as podocin, and failed to demonstrate long term functional expression of such genes.
• the present invention aims to reverse the NS phenotype and correct podocyte- associated kidney dysfunction in patients with monogenic forms of NS by administering AAV gene therapy expressing a NS-associated transgene under the control of a podocyte-specific promoter.
• the present invention provides an adeno-associated virus (AAV) vector gene therapy for use in treating a monogenic form of nephrotic syndrome, wherein the AAV vector comprises: a NS-associated transgene; and minimal nephrin promoter NPHS1 or podocin promoter NPHS2.
• AAV adeno-associated virus
• the gene therapy vector can reverse the NS phenotype and correct podocyte-associated kidney dysfunction in patients with monogenic forms of NS.
• Suitable AAV serotypes for use in the vector include 2/9, LK03 and 3B.
• the AAV 2/9 serotype has shown significant tropism for newborn and adult mouse kidney, localising to the glomeruli and tubules (Luo et al., 2011; Picconi et al., 2014; Schievenbusch et al., 2010), and AAV2/9 vector combined with renal vein injection has been shown to be suitable for kidney-targeted gene delivery (Rocca at al., 2014).
• AAV 2/9 is therefore one suitable vector for use in the gene therapy of the present invention.
• Synthetic AAV capsids such as LK03 can also be suitable vectors for use in the gene therapy of the present invention.
• This vector has been shown to transduce human primary hepatocytes at high efficiency in vitro and in vivo. However, until now it has not been utilised in kidney-targeted gene delivery.
• the present inventors have demonstrated herein that AAV-LK03 vectors can achieve high transduction of close to 100% in human podocytes in vitro and can be used to transduce podocytes specifically in vitro.
• the AAV-LK03 cap sequence consists of fragments from seven different wild-type serotypes (AAV1, 2, 3B, 4, 6, 8, 9), although AAV-3B represents 97.7% of the cap gene sequence and 98.9% of the amino acid sequence.
• AAV-3B is also known for its human hepatocyte tropism is another a suitable vector for use in the gene therapy of the present invention. To date it has not been utilised in kidney- targeted gene delivery.
• the NS-associated transgene used in the gene therapy is a gene associated with a monogenic form of NS and expressed in podocytes, and which encodes a protein of about 833 amino acids or less. This size limitation allows the NS-associated transgene to fit into the gene therapy vector of the present invention.
• Suitable NS-associated transgenes include NPHS2 ; ADCK4 ; ALG1 ; ARHGAP24 ; ARGHDIA CD151 - CD2AP ; COQ2 ; COQ6 ; DGKE ; E2F3; EMP2) KANK2 ; LAGE3) LMNA LMX1B ; MAFB ; NUP85) NUP93 ; NXF5) OSGEP ; PAX ; PDSS2) PM M2) PODXL) SCARB2) SGPL1 ) Smad7) TP53RK) TPRKB) VDR; WDR73) WT1 ) ZMPSTE24) and APOL1.
• the NS-associated transgene may be an SRNS- associated transgene such as ADCK4) CD2AP) DGKE) EMP2) NPHS2) NUP86) NUP93) SGPL1 ; WDR73) or I/I/P .
• the NS-associated transgene is NPHS2, which encodes podocin.
• NPHS2 encodes podocin.
• Figure 6 One example of a suitable human NPHS2 transgene cDNA sequence is shown in Figure 6.
• the transgene species is preferably matched to the patient species. For example, when treating a human patient one would typically use a human transgene.
• the transgene may be naturally occurring, e.g. wild-type, or it may be recombinant.
• the transgene is typically included in the gene therapy vector as a cDNA sequence.
• NPHS1 or podocin promoter NPHS2 allows the gene therapy vector to be targeted specifically to podocytes (Moeller et al., 2002; Picconi et al., 2014). This enables transgene expression to be specifically targeted to podocytes in the glomerular basement membrane of the kidney and minimises off-target expression. As podocytes are terminally differentiated and non-dividing cells they can be targeted for stable expression of the transgene and reduce or avoid any risk of vector dilution effect.
• the promoter is NPHS1.
• NPHS1 One example of a suitable DNA sequence for the NPHS1 promoter is shown in Figure 5.
• the species of the promotor is preferably matched to the patient species.
• the AAV vector may additionally comprise a Woodchuck hepatitis post- transcriptional regulatory element (WPRE).
• WPRE is a DNA sequence that, when transcribed, creates a tertiary structure enhancing expression. Inclusion of WPRE may increase expression of the transgene delivered by the vector.
• the WPRE sequence may be mutated to reduce oncogenicity without significant loss of RNA enhancement activity (Schambach et a/., 2005, incorporated herein by reference).
• a suitable WPRE sequence is shown in Figure 7.
• the NS-associated transgene may comprise a hemagglutinin (HA) tag.
• HA can be used as an epitope tag and has been shown not to interfere with bioactivity or biodistribution of proteins to which it has been added.
• the HA tag can facilitate detection, isolation, and purification of the transgene.
• the AAV vector may additionally comprise a Kozak sequence between the promoter and the podocin transgene. The Kozak sequence is known to play a major role in the initiation of the translation process and can therefore enhance expression of the podocin transgene.
• the AAV vector may additionally comprise a polyadenylation signal, such as bovine growth hormone (bGH) polyadenylation signal, e.g. as shown in Figure 8.
• bGH bovine growth hormone
• Polyadenylation is the addition of a poly(A) tail to a messenger RNA.
• the poly(A) tail consists of multiple adenosine monophosphates; in other words, it is a stretch of RNA that has only adenine bases.
• the poly(A) tail is important for the nuclear export, translation, and stability of mRNA. Inclusion of a polyadenylation signal can therefore enhance expression of the podocin transgene.
• the AAV vector gene therapy additionally typically comprises Inverted Terminal Repeat (ITR) sequences at either end of the vector.
• ITR Inverted Terminal Repeat
• the vector structure may be, in order: ITR - promotor - transgene (with optional HA tag) - optional WRPE - polyadenylation signal - ITR.
• the gene therapy vector of the present invention can therefore be used to treat or manage monogenic forms of NS in a patient.
• patient as used herein may include any mammal, including a human.
• the patient may be an adult or a paediatric patient, such as a neonate or an infant.
• the patient may be a paediatric patient between the ages of about 1 and about 16 years old.
• the patient is suffering from a monogenic form of NS.
• the NS is caused by a mutation in one gene.
• mutation is in a gene expressed in podocytes.
• the NS may be SRNS caused by a mutation in NPHS2, which encodes podocin.
• the monogenic form of NS may be caused by one or mutations in any one of ADCK4) ALG1 ; ARHGAP24 ; ARGHDIA ; CD151 ; CD2AP- COQ2- COQ6 ; DGKE ; E2F3 ; EMP2) KANK2 ; LAGE3 ; LMNA ; LMX1B ; MAFB ; NUP85) NUP93) NXF5 ; OSGEP) PAX2 ; PDSS2) PM M2) PODXL) SCARB2) SGPL1 ) Smad7) TP53RK) TPRKB) VDR) WDR73) WT1 ) ZMPSTE24) or APOL1.
• ADCK4 ALG1 ; ARHGAP24 ; ARGHDIA ; CD151 ; CD2AP- COQ2- COQ6 ; DGKE ; E2F3 ; EMP2) KANK2 ; LAGE3 ; LMNA ; LMX1B ; MAFB ; N
• the monogenic form of NS may be a monogenic form of SRNS caused by one or more mutations in any one of ADCK4) CD2AP) DGKE) EMP2) NPHS2) NUP86) NUP93) SGPL1 ) WDR73) or WTl .
• the genetic mutation causing the SRNS may be an NPHS2 mutation affecting podocin expression, such as one or more of those listed in Table A below.
• Table A non-exhaustive list of podocin ( NPHS2 ) mutations.
• the genetic mutation may be p.Argl38Gln, also known as R138Q.
• R138Q is the most common podocin mutation in children with SRNS in Caucasian populations. The mutation causes endoplasmic reticulum retention of podocin, which prevents it from reaching the slit diaphragm and interacting with other important slit diaphragm proteins to form a functional filtration barrier.
• the NPHS2 mutations are all affecting the same gene any combination of these mutations could be treated by an AAV gene therapy vector of the present invention including an NPHS2 transgene.
• the patient may have a p.Argl38Gln mutation and may also have one or more of the other mutations identified in Table A above.
• the presence or absence of a monogenic form of NS can be determined by laboratory testing, such as that available from Bristol Genetics Laboratories, Bristol, UK. Typically, genetic testing can be performed by analysis of a blood sample obtained from the patient.
• the AAV vector gene therapy may be administered systemically, such as by intravenous injection.
• the AAV vector gene therapy may be administered by injection into the renal artery.
• the AAV vector gene therapy may be administered by retrograde administration, e.g. via the ureters using a urinary catheter.
• the gene therapy may be administered as a single dose, in other words, subsequent doses of the vector may not be needed.
• vector used in a first dose may comprise AAV-LK03 or AAV-3B whereas the vector used in a subsequent dose may comprise AAV 2/9.
• the gene therapy may be administered in combination with temporary immunosuppression of the patient, e.g. by administering the gene therapy at the same time as, or following treatment with, oral steroids. Immunosuppression may be desirable before and/or during gene therapy treatment to suppress the patient's immune response to the vector.
• the AAV capsid is present only transiently in the transduced cell as it is not encoded by the vector.
• the capsid is therefore gradually degraded and cleared, meaning that a short-term immunomodulatory regimen that blocks the immune response to the capsid until capsid sequences are cleared from the transduced cells can allow long-term expression of the transgene. Immunosuppression may therefore be desirable for a period of about six weeks following administration of the gene therapy.
• the AAV vector gene therapy may be administered in the form of a pharmaceutical composition.
• the AAV vector gene therapy may be combined with one or more pharmaceutically acceptable carriers or excipients.
• a suitable pharmaceutical composition is preferably sterile.
• Figure 1 shows that AAV 2/9 administered by tail vein injection transduces the kidney and expresses HA-tagged podocin in the podocyte.
• Figure 2 shows that tail vein injection of AAV 2/9 expressing wild-type podocin under a podocyte-specific promoter ameliorates proteinuria in the conditional podocin knock-out mouse model (iPod NPHS2 fl/fl )
• A) Urinary albumi creatinine ratio of mice injected with AAV 2/9 mNPHSl.mpod versus AAV 2/9 hNPHSl.mpod versus saline (n 9 in each group, **p ⁇ 0.01 ***p ⁇ 0.001).
• the saline group showed proteinuria from day 14 onwards and showed a large amount of albumin while the vector treated groups showed later onset of albuminuria and milder albuminuria.
• AAV LK03 shows efficient transduction of human podocytes in vitro with the minimal human nephrin promoter.
• A, C, E immunofluorescence demonstrating transduction of human podocytes (Pod), glomerular endothelial cells (GEnC) and proximal tubule epithelial cells (PTEC) by AAV LK03 CMV GFP, with only expression of GFP in podocytes when using the minimal nephrin promoter
• AAV LK03 hNPHSl GFP AAV LK03 hNPHSl GFP.
• B Western blot demonstrating GFP expression in podocytes only when using the minimal human nephrin promoter using AAV LK03.
• D) Flow cytometry demonstrating highly efficient transduction of podocytes using AAV LK03 CMV GFP, and confirming the GFP expression using the minimal nephrin promoter was only seen in podocytes.
• FIG. 4 AAV LK03 expressing wild type human podocin shows functional rescue in the mutant podocin R138Q podocyte cell line.
• Figure 5 shows an example DNA sequence for the minimal human nephrin promoter (NPHS1).
• Figure 6 shows an example cDNA sequence for a human podocin transgene.
• Figure 7 shows an example DNA sequence for a WPRE sequence.
• Figure 8 shows an example DNA sequence for a bGH poly(A) signal sequence.
• Figure 9 shows human podocytes transduced with either HAVDR (A) or HASmad7 (B) using AAV LK03 with the minimal human nephrin promoter.
• Vector production We prepared pAV.hNPHSl.mpodHA.WPRE.bGH, pAV.mNPHSl.mpodHA.WPRE.bGH and pAV.hNPHSl.hpodHA.WPRE.bGH ( Figure 1A) pAV.mNPHSl.hHAVDR.WPRE.bGH and pAV.mNPHSl.hHASmad7.WPRE.bGH in our laboratory from a CMV eGFP L22Y pUC-AV2 construct (kind gift of Amit Nathwani) using human ( Figure 6) and mouse (sequence not shown) podocin cDNA (Origene, Herford, Germany) and human VDR and Smad7 cDNA.
• Human embryonic kidney 293T cells were transfected with a capsid plasmid (pAAV9 from Penn Vector Core, pAAV LK03 was the kind gift of Mark Kay), a helper plasmid with adenoviral genes and the transgene plasmid using polyethyleneimine. Cells and supernatant were harvested at 72 hours post-transfection. Cells underwent 5 freeze-thaw cycles, while the supernatant underwent PEG precipitation (8% PEG 0.5N NaCI). These were combined and incubated with 0.25% sodium deoxycholic acid and 70units/ml Benzonase for 30 minutes at 37°C. The vector was purified by iodixanol gradient ultracentrifugation, and subsequently concentrated in PBS. Vectors were titrated by qPCR using the standard curve method using the following primers:
• NPHS2 fl0X/fl0X mice (kind gift of Corinne Antignac, INSERM U983, Paris) were bred with NPHS2-rtTA/ Tet-On Cre mice to generate offspring with NPHS2-rtTA/ Tet-On Cre/ NPHS2 fl0X/fl0X . These mice develop a podocyte-specific knockout of podocin when exposed to doxycycline. These will be called iPod NPHS2 fl/fl from hereon.
• mice were on a mixed background and equal numbers of each sex were used. Mice were administered AAV via tail vein injection at 8 weeks of age. (Figure IB) 10 to 14 days later, mice were provided with drinking water supplemented with doxycycline 2mg/ml and 5% sucrose for 3 weeks. Urine was taken weekly. Mice were culled by Schedule 1 methods at 6 weeks post initiation of doxycycline. A small number of mice were kept beyond 6 weeks to test for effect on survival. All mice were re-genotyped from tissue taken at death. Cell Culture
• Conditionally immortalised human podocytes were cultured in RPMI with L- glutamine and NaHC03 with 10% Fetal Bovine Serum (Sigma Aldrich, Gillingham, UK).
• Conditionally immortalised human glomerular endothelial cells were cultured in EBMTM-2 Endothelial Cell Growth Basal Medium-2 supplemented with EGMTM-2 Endothelial Cell Growth Medium-2 BulletKitTM (Lonza, Basel, Switzerland).
• Immortalised proximal tubule epithelial cells (ATCC, Teddington, UK) (PTEC) were cultured in DMEM/F12 supplemented with Insulin, Transferrin and Selenium, Hydrocortisone and 10% FBS.
• Cells were transduced with AAV at a MOI of 5 x 10 5 .
• GFP expression cells were used at 5-7 days post transduction to allow comparisons across different cell lines.
• podocin, VDR and Smad7 expression cells were used at 10-14 days post transduction when podocytes are maximally differentiated.
• DNA was extracted using DNeasy Blood and Tissue Kit (Qiagen, Manchester, UK) from mouse kidney cortex. AAV DNA was detected using the primers above for viral titration and normalised against mouse beta-actin.
• Cells were fixed with either 4% PFA and or ice cold methanol, incubated for 5 minutes with 0.03M glycine, permeabilised with 0.3% Triton then blocked with 3% BSA.
• Primary antibodies were mouse HA.11 Epitope Tag Antibody (Biolegend, San Diego, USA), mouse anti-GFP (Roche, Basel, Switzerland), rabbit anti-Calnexin (Merck Millipore, Darmstadt, Germany) and rabbit anti-Caveolin 1 (Cell Signaling, Danvers, USA).
• AlexaFluor 488 donkey anti-mouse AlexaFluor 488 donkey anti-rabbit
• AlexaFluor 488 goat-anti guinea pig AlexaFluor 555 goat anti rabbit and AlexaFluor 633 goat anti-rat
• AlexaFluor 633 Phalloidin Invitrogen, Thermo Fisher Scientific, Waltham, USA. Sections were counterstained with DAPI and mounted with Mowiol.
• Live cells were stained with propidium iodide and only live single cells were included in the analysis.
• Flow cytometry was carried out on the NovoCyte Flow Cytometer.
• Cells were trypsinised and resuspended at 10 5 /ml and allowed to recover for 10 minutes before plating 50pl of cells diluted 1 in 2 with PBS in a 96 well plate. Technical triplicates were used. Cells were left to adhere for about 1 hour at 37°C. Cells were washed with PBS to wash away non adherent cells, then fixed with 4% PFA for 20 minutes. Cells were washed with distilled water then stained with 0.1% crystal violet in 2% ethanol for 60 minutes at room temperature. Cells were washed and incubated with 10% acetic acid on a shaker for 5 minutes. Absorbance was measured at 570nm and results were normalised against the wild type cell line transduced with AAV LK03 CMV GFP.
• Albumin levels were measured using a mouse albumin ELISA kit (Bethyl Laboratories Inc, Montgomery, USA) and Creatinine levels were measured on the Konelab Prime 60i Analyzer.
• Mouse plasma was processed either using the Konelab Prime 60i analyser or the Roche Cobas system with reagents and protocols supplied by the manufacturer.
• Tail vein injection of AAV serotype 9 demonstrates transduction of kidney cells and expression in the podocyte
• AAV2/9 expressing wild type podocin reduces albuminuria in iPod NPHS2 fi/fl mice
• Vector treated groups showed a reduction in urinary albumin :creatinine ratio (ACR) ( Figure 2A, 2B).
• mice in AAV 2/9 hNPHSl.mpod group and 1 of 9 mice in AAV 2/9 mNPHSl.mpod group had urinary ACRs of less than 30mg/mmol at day 42.
• mice in vector treated groups showed an improvement, there was a large degree of variation within the groups which we hypothesised might be attributable to amount of vector that reached the kidney after a systemic injection.
• AAV2/9 expressing wild type podocin partially rescues the phenotype in iPod NPHS2 fl/fl mice
• AAV LK03 transduces human podocytes efficiently in vitro with the minimal human nephrin promoter
• AAV LK03 with CMV GFP and AAV LK03 hNPHSl GFP were used to transduce human podocytes, glomerular endothelial cells and proximal tubular epithelial cells at a MOI of 5x10 s .
• AAV 2/9 has been the serotype which has seen the best transduction in kidney cells in vivo in rodent kidneys, we tested the expression of AAV 2/9 CMV GFP on human kidney cell lines.
• AAV LK03 with AAV LK03 hNPHSl HAVDR and AAV LK03 hNPHSl hSmad7 were used to transduce human podocytes showing good expression of both proteins (Figure 9).
• AAV LK03 expressing human podocin under the minimal nephrin promoter shows functional rescue in mutant podocin R138Q podocyte cell line
• the R138Q podocin mutant results in mislocalisation of podocin from the plasma membrane to the endoplasmic reticulum.
• the mutant podocin R138Q podocyte cell line was acquired from a patient kidney and conditionally immortalised using temperature sensitive SV40 T antigen.
• AAV LK03 hNPHSl hpod transduces R138Q podocytes and expresses HA-tagged podocin ( Figure 4A, 4B).
• HA-tagged podocin is seen at the plasma membrane on confocal microscopy and colocalises with Caveolin-1, a lipid raft protein, as seen on TIRF microscopy ( Figure 4B, 4E).
• Untransduced R138Q podocytes do not show any podocin expression at the plasma membrane ( Figure 4B).
• HA-tagged podocin does not colocalise with Calnexin, an endoplasmic reticulum marker ( Figure 4D).
• Podocytes show a decrease or increase in adhesion in diseased states. Previous work in our laboratory has shown that the R138Q mutation causes a decrease in podocyte adhesion. AAV transduction causes a decrease in podocyte adhesion but the R138Q podocytes still show reduced adhesion compared to wild type podocytes, and transduction with AAV LK03 hNPHSl hpod results in the rescue of the adhesional function of R138Q podocytes ( Figure 4C).
• AAV LK03 has shown high transduction of close to 100% in human podocytes in vitro, which is reduced to 72.3% when using the minimal human nephrin promoter. We have shown that we can use this serotype to transduce podocytes specifically in vitro, and that expression of wild type podocin in R138Q mutant podocytes show functional rescue. Using AAV LK03 has implications on translation as such effective transduction of human podocytes might enable a significant reduction in effective dose in humans.
• a recent UK study has shown low anti AAV LK03 neutralising antibody seroprevalence of 23%, with a nadir in late childhood (Perocheau, D. P. et al.), which makes this particular serotype a promising candidate for translational studies.
• rAAV9 combined with renal vein injection is optimal for kidney-targeted gene delivery: conclusion of a comparative study. Gene therapy, 21, 618-628.
• VAN DER WOUDEN E. A., SANDOVICI, M., HENNING, R. H., DE ZEEUW, D. & DEELMAN, L. E. 2004. Approaches and methods in gene therapy for kidney disease. J Pharmacol Toxicol Methods, 50, 13-24.
• [SEQ ID NO: l] shows the ITR Forward primer.
• [SEQ ID NO: 2] shows the ITR Reverse primer.
• [SEQ ID NO: 3] shows the DNA sequence of the ITR probe FAM-5'- CACTCCCTCTCTGCGCGCTCG-3'-TAMRA.
• [SEQ ID NO:4] shows the example DNA sequence for the minimal human nephrin promoter (NPHS1) as shown in Figure 5.
• [SEQ ID NO: 5] shows the example cDNA sequence for the human podocin transgene shown in Figure 6.
• [SEQ ID NO:6] shows the example DNA sequence for the WPRE sequence shown in Figure 7.
• [SEQ ID NO:7] shows the example DNA sequence for the bGH poly(A) signal sequence shown in Figure 8.

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